Structural mechanism of BRD4-NUT and p300 bipartite interaction in propagating aberrant gene transcription in chromatin in NUT carcinoma

BRD4-NUT, a driver fusion mutant in rare and highly aggressive NUT carcinoma, acts in aberrant transcription of anti-differentiation genes by recruiting histone acetyltransferase (HAT) p300 and promoting p300-driven histone hyperacetylation and nuclear condensation in chromatin. However, the molecular basis of how BRD4-NUT recruits and activates p300 remains elusive. Here, we report that BRD4-NUT contains two transactivation domains (TADs) in NUT that bind to the TAZ2 domain in p300. Our NMR structures reveal that NUT TADs adopt amphipathic helices when bound to the four-helical bundle TAZ2 domain. The NUT protein forms liquid-like droplets in-vitro that are enhanced by TAZ2 binding in 1:2 stoichiometry. The TAD/TAZ2 bipartite binding in BRD4-NUT/p300 triggers allosteric activation of p300 and acetylation-driven liquid-like condensation on chromatin that comprise histone H3 lysine 27 and 18 acetylation and transcription proteins BRD4L/S, CDK9, MED1, and RNA polymerase II. The BRD4-NUT/p300 chromatin condensation is key for activating transcription of pro-proliferation genes such as ALX1, resulting ALX1/Snail signaling and epithelial-to-mesenchymal transition. Our study provides a previously underappreciated structural mechanism illuminating BRD4-NUT’s bipartite p300 recruitment and activation in NUT carcinoma that nucleates a feed-forward loop for propagating histone hyperacetylation and chromatin condensation to sustain aberrant anti-differentiation gene transcription and perpetual tumor cell growth.

fusions include dysregulation of parent gene expression and chromosome aberrations by fusion genes 1,7 . However, for solid tumors (mesenchymal and epithelial), disease-related molecular signatures of fusion genes and their encoded proteins remain poorly understood 8 .
Nuclear protein in testis (NUT) carcinoma (NC, or NUT-midline carcinoma, NMC) is a rare and one of the most aggressive solid tumors with a median survival of 6.5 months 9 . It often arises in the midline regions (head, neck, lungs, or thorax), and affects primarily adolescents and young adults 10 . The genetic hallmark of NC is a rearrangement of NUT on chromosome 15q14 that is fused with BRD4 (but in some cases BRD3, NSD3, or other genes), creating a fusion gene that encodes BRD4-NUT fusion protein (Fig. 1a) 11,12 . Unlike many gene fusions that directly alter gene expression, BRD4-NUT acts as a transcription co-activator. It binds to lysine-acetylated histones through BRD4 tandem bromodomains (BrDs) and recruits histone acetyltransferase (HAT) p300/CBP to chromatin through the NUT moiety 13,14 (Fig. 1a). The p300/CBP recruitment induces excessive histone H3 lysine 27 acetylation (hyper-acetylation; H3K27ac) and creates unusually large topological domains (aka megadomains) in chromatin that enable aberrant gene transcription by sequestering transcriptional activators away from pro-differentiation genes [12][13][14][15][16][17] and activating proliferation and anti-differentiation genes [16][17][18][19][20][21] , resulting in perpetual tumor cell growth. Notably, p300/CBP recruitment does not always correlate with chromatin acetylation including H3K27ac and gene activation 22,23 . Therefore, it is imperative to examine the mechanism that regulates BRD4-NUT recruitment of p300/CBP and the resulting activation of p300/CBP HAT activity in cellular progression of NC.
The histone acetyltransferase p300/CBP, contains both structured and disordered domains, including nuclear receptor interaction domain (NRID, disordered in free state), transcription adapter zinc binding domains (TAZ1, TAZ2), and domains such as KIX, BrD, PHD, HAT, and ZZ ( Fig. 1a) [24][25][26][27] . The TAZ2 domain is an adaptor protein module that enables p300 to nucleate the assembly of diverse transcription factors (TFs) into multicomponent co-activator complexes including p53, IRF3, STAT3, and MEF2 [28][29][30][31] . As shown previously 27,31,32 , p300 normally is in a dimeric and autoinhibitory state for its HAT activity, and gets activated upon binding to phosphorylated and dimerized transcription factors (IRF3 and STAT1) on chromatin. The p300/transcription factor binding-induced p300 activation is shown by p300 trans-autoacetylation and its substrate acetylation of histone H3K18 and H3K27 in chromatin 31 . Although it has been reported that the F1c region in NUT of BRD4-NUT interacts with the TAZ2 domain of p300 ( Fig. 1a) 13 , the structural and molecular details of the F1c/TAZ2 interaction and its relation to p300 activation, histone hyperacetylation, and aberrant gene transcription for NUT carcinoma cell proliferation have remained elusive 13 .
In this study, we report that BRD4-NUT contains two transactivation domains (TADs) in NUT that bind to the four-helical bundle TAZ2 domain of p300. The BRD4-NUT/p300 bipartite binding activates p300 HAT and results in histone H3 lysine 27 and 18 hyperactivation, leading to formation and spread of BRD4-NUT/p300 nuclear condensation on chromatin that comprise key transcription proteins BRD4L/S, CDK9, MED1, and RNA polymerase II. We further show that the BRD4-NUT/p300 chromatin condensation is important for transcriptional activation of pro-proliferation genes such as ALX1, a highly expressed oncogene in NUT carcinoma cells, and resulting ALX1/Snail signaling and epithelial-to-mesenchymal transition.
We then used synthetic peptides to define the F1c binding motifs (Supplementary Table 1). Addition of the F1c peptide (residues 403-418) caused characteristic but less chemical shift perturbations in the HSQC spectrum of the 15 Fig. 2a), which is consistent with their binding affinity, i.e., K d~1 15 μM for residues 403-418 vs. K d~3 2 μM for residues 377-418 (Fig. 1f, Supplementary Fig. 2a). These results indicate that the 403-418 peptide resembles the essential binding motif for the 377-418 sequence. Another F1c peptide (residues 435-454) did not exhibit much binding to the TAZ2 domain as shown by HSQC spectra and weak affinity (K d~9 58 μM) ( Supplementary Fig. 2b). Accordingly, we engineered a fusion TAZ2-NUT protein by joining the TAZ2 domain (residues 1723-1812) to NUT (residues 419-470) with a flexible sixresidue Gly-Ser repeat sequence (Fig. 1c). The NMR chemical shift differences between 15 N-TAZ2-NUT and 15 N-TAZ2 (free state) are nearly identical to those of 15 N-TAZ2 upon addition of NUT (residues 419-470) ( Fig. 1h right panel, Supplementary Fig. 2c). Therefore, our engineered TAZ2-NUT protein represents an accurate binding model of the TAZ2 and NUT (residues 419-470) complex, allowing us to determine the high-resolution structure of this complex.
In the TAZ2-NUT TAD2 structure, the fused NUT sequence of residues 433-460 forms two anti-parallel α-helices. The first α-helix (α1', residues 433-440) is in a similar position as the TAD1 peptide (residues 403-418), whereas the second α-helix (α2', residues 448-460) flips back to interact with the first α-helix (α1', residues 433-440) and the α2-helix of TAZ2 protein surface (Fig. 2b vs. 2a). For example, side chains of F435, L436, L439, L440 from the α1'-helix are situated in the TAZ2 binding pocket. P448, L449, L451, I452, and L455 from the α2'-helix interact with TAZ2 residues on the α2 and α3 helices and the TAD1 α1'-helix (Fig. 2b). Side chains of preceding residues A429, V430, and I431 participate in hydrophobic interactions with a Interaction between p300 TAZ2 domain and F1c peptide (TAD1, residues 403-418). Left: Ribbon depiction of the lowest energy structure of the complex; Middle: Expanded diagram of key inter-molecular interactions from the α-helix F1c peptide to the hydrophobic binding core of the TAZ2 domain surrounded by α1, α2, and α3 helices. TAZ2 domain residues involved in peptide binding are labeled and colored in light-green, the peptide elements are colored in blue; Right: Surface representation of the TAZ2 domain depicting its recognition to the F1c peptide in the same orientation as (Middle). b Structure of TAZ2-NUT fusion protein containing the NUT sequence (TAD2, residues 419-470). Left: Ribbon depiction of the lowest energy structure of the fusion protein (TAZ2 in light-blue and NUT sequence in yellow); Middle: The binding core depiction of residue side-chains of TAZ2 in α1, α2, and α3 helices for hydrophobic and electrostatic interactions to the residues in the F1c sequence. Right: Surface representation of interaction positions of TAZ2-NUT fusion in the same orientation as (Middle); c The ribbon diagram of CBP TAZ2/ p53 TAD2 complex. d Superposition of NUT TAD2 sequence (residues 419-470, in yellow) in the TAZ2-NUT fusion protein with NUT TAD1 sequence (residues 403-418, in blue), and p53 TAD2 (residues 35-59, in orange, 2MZD) peptides. e Sequence alignment of NUT TAD1 (residues 405-414), NUT TAD2 (residues 430-458), p53 TAD2, and p63 TAD. The conserved proline amino acid is colored in red, hydrophobic residues in blue. The binding motif common to these sequences is diagramed as, Φ represents a hydrophobic amino acid and X represents any amino acid. residues located on α1 and α3 helices of TAZ2. Together, they establish more extensive contact with the TAZ2 surface (Fig. 2b). Side chain of D447 of NUT also forms electrostatic interactions with R1763 of TAZ2, and the carbonyl group of D438 backbone is within hydrogen-bonding distance with the side chain of K1760 of TAZ2 domain (Fig. 2b). Both NUT TAD1 and TAD2 sequences adopt a similar conformation on the TAZ2 domain as compared to the p53-TAD2 structure (Fig. 2c, d), indicating that TAZ2 functions as an adaptor to recruit transcription proteins. Alignment of NUT TAD1 and TAD2 residues 403-418 and 428-445 with p53-TAD2 or p63-TAD peptides reveals consensus binding sequence of ΦΦxPxΦΦxxΦΦ (where P represents proline, Φ is a hydrophobic amino acid, and x any amino acid; Fig. 2e) 36 .

NUT liquid-like condensation is enhanced by the p300 TAZ2 incorporation
Recent studies from us and others showed that protein IDRs mediate liquid-like condensation in the nuclear assembly for gene transcription [37][38][39] . We investigated the condensation capability of NUT IDRs in the constructs with an EGFP tag (A206K) (outlined in Fig. 1c). Purified recombinant EGFP-NUT (residues 300-700) protein formed spherical droplets in a concentration-dependent manner (Fig. 3a). Fluorescence microscopy of the opaque EGFP-NUT solutions revealed that the GFP-positive droplets appeared stable as salt concentration increased to examine the contribution of electrostatic interactions (Fig. 3b). The micron-sized droplets fused, coalesced and dripped (Fig. 3c), but dispersed following the treatment of 1,6-hexanediol that is known to perturb weakly hydrophobic interactions ( Supplementary Fig. 4a). We next prepared a shorter EGFP-F1c segment (residues 346-592), which consisted of key α-helical binding motifs but with truncated IDRs as compared to EGFP-NUT protein (residues 300-700) (Fig. 1c). Microscopic images showed that this EGFP-F1c protein produced smaller and less abundant liquid droplets, which had the higher threshold concentration (10 μM) and were more sensitive to salt than EGFP-NUT droplets (0.  Quantitative data are shown as mean +/− S.D. (n = minimum of four independent samples for each condition). p-values for each indicated comparison are two-tailed unpaired and derived from Student's t-test without adjustment for multiple comparisons. e Supernatant (S) and pelleted (P) fractions from droplet spin down experiments were run in western blots to assess the time-dependent (0-24 h of phase separation) formation of heat-, reducing-and SDS-stable EGFP-NUT/Cherry-TAZ2 oligomers. Note that some high molecular weight protein complexes (HMW multimers) appeared shortly after addition of Cherry-TAZ2. Data represent mean +/ − SEM from three independent experiments. p-values for each indicated comparison are two-tailed unpaired and derived from Student's t-test without adjustment for multiple comparisons.
in the NUT protein for promoting droplet formation. Both EGFP-NUT and EGFP-F1c droplets can form in the absence of EGFP ( Supplementary Fig. 4d).
We next investigated how p300 TAZ2-binding to NUT may facilitate liquid-like condensation. Addition of Cherry-TAZ2 could incorporate and thus concentrate the droplets of EGFP-NUT (Fig. 3d vs. Fig. 3a) or EGFP-F1c ( Supplementary Fig. 4b, e), while equivalent solutions with Cherry-TAZ2 alone remained homogenous (Fig. 3d). Notably, the droplet sizes and numbers were significantly enhanced after the NUT/TAZ2 molar ratio reached above 1:2 stoichiometry (Fig. 3d), suggesting that NUT bipartite motifs act as binding scaffolds to incorporate TAZ2 domain of p300 into the complex condensation 37 . We isolated droplets from the solution phase by sedimentation and determined the levels of NUT and TAZ2 in the pellet fraction by immunoblotting. Co-addition of EGFP-NUT and Cherry-TAZ2 resulted in high molecular weight (HMW) oligomerization in a time-dependent manner, indicating that their incorporation leads to enrichment of stable oligomers (Fig. 3e). Notably, the EGFP-NUT/Cherry-TAZ2 droplets were still sensitive to 1,6-hexanediol treatment (Supplementary Fig. 4f).
We further observed that transcription machinery components Mediator subunit MED1 and CDK9 formed puncta that partially overlapped with EGFP-BRD4-NUT-WT puncta ( Supplementary Fig. 6a), indicating that the transcription components are drawn within proximity but may not totally colocalize with BRD4-NUT puncta. Similarly, RNA polymerase II (Pol II) phosphorylated at serine 2 (S2P) or serine 5 (S5P) was shown in nuclear puncta, which were in partial colocalization with BRD4-NUT puncta. These results agree with the previously reported limited association between BRD4-NUT and transcription components 13,15 . Notably, transfection of the deletion mutant BRD4-NUT-ΔF1c largely abolished condensation and colocalization with MED1, CDK9, RNA Pol II S2P and S5P (Supplementary Fig. 6a, b). These results indicate that, in addition to BRD4 BrD acetyl-lysine binding, BRD4-NUT/p300 bipartite association and p300 activation are required to nucleate BRD4-NUT co-condensation with transcription proteins, and propagate histone hyperacetylation, which negatively 13,15,16 or positively 16,[18][19][20] alters gene transcription in chromatin. The functional detail between BRD4-NUT and RNA Pol II in NC cells warrants further investigation 21 .

BRD4-NUT/p300 bipartite interaction activates its HAT activity and histone acetylation
We observed that BRD4-NUT-WT ectopically expressed in HEK293Tcells activates p300 HAT activity as reflected by markedly increased levels of p300 acetylation (p300-Kac), and p300 histone H3 substrates H3K18ac and H3K27ac 13 , whereas the single TAD1 or TAD2 deletion or double deletion (ΔF1c) mutant showed nearly complete loss in their ability to activate p300 (Fig. 5a, Supplementary Fig. 7a). Further, in an in-vitro HAT assay with nuclear extracts of HEK293T cells overexpressing EGFP-BRD4-NUT-WT or the TAD deletion mutants, we confirmed that BRD4-NUT-WT increased acetylation levels of endogenous p300 (p300-Kac) and histone H3 (H3K27ac, H3K18ac, and H3K56ac), whereas neither of the single nor double TAD1/2 deletion mutants yielded any noticeable enhancement of p300 or histone H3 acetylation as compared to the empty vector control (Fig. 5b).    Collectively, these results demonstrate that BRD4-NUT bipartite interaction with p300 is required for p300 HAT trans-activation and histone H3 hyper-acetylation in chromatin. Given that many transcription factors including p53 directly interact with p300/CBP through their TAD domains 42 , we investigated whether such tandem TADs form bipartite interactions with p300 and activate the protein. We found that TAD1 and/or TAD2 deletion in p53 reduced its interaction with p300 ( Supplementary Fig. 7c), and that the wild-type EGFP-p53-WT ectopically expressed in HEK293T cells, but not its TAD1/2 deletion mutants (ΔTAD1, -ΔTAD2, or -ΔTAD1/2), increased levels of p300-Kac, H3K18ac, H3K27ac, and p53-Kac in both cellular and in-vitro acetylation assays ( Supplementary Fig. 7d, e). These results confirm that p53 TADs form bipartite interactions with p300, resulting in p300 and p53 activation and histone acetylation.
We found from analyzing available RNA-seq data that ALX1 was significantly overexpressed in NC lung cells HCC2429 and PER-403 in comparison to non-NC lung cells HCC1359, H661 and A549, whereas its expression was relatively low in NC thymus cells TC-797 (Fig. 6a). Further, knockdown of BRD4-NUT substantially downregulated the ALX1 mRNA levels in PER-403 cells but to a lesser extent in TC-797 cells (Fig. 6b) likely due to low basal ALX1 level. These results suggest that BRD4-NUT regulates tissue-specific transcription of ALX1 in NC types.
To examine how BRD4-NUT/p300 facilitates ALX1 gene expression, we transfected wild-type Flag-BRD4-NUT-WT or TAD1/2 deletion mutant Flag-BRD4-NUT-ΔF1c in HEK293T cells. Transfection of Flag-BRD4-NUT-WT stimulated transcriptional expression of ALX1, SOX2, and TP63 (Fig. 6c), which are all important in Snail expression and squamous cancer progression 43,46 . In agreement with the results of BRD4-NUT knockdown in NC cells 47 , expression of the TAD1/2 deletion mutant BRD4-NUT-ΔF1c markedly suppressed activation of ALX1 transcription (Fig. 6c), indicating the direct role of TAD1/2 of BRD4-NUT in control of ALX1 transcription. Notably, this TAD1/2 deletion mutant had varying inhibitory effects on SOX2 and TP63 expression, suggesting that additional factors may be involved in BRD4-NUT modulation of transcriptional activation of these genes ( Fig. 6c; Supplementary Fig. 8a, b). Our ChIP-qPCR further confirmed that transiently expressed Flag-BRD4-NUT-WT or Flag-BRD4-NUT-ΔF1c mutant was present at the ALX1 promoter site in HEK293T cells, and that the former correlates to enrichment levels of H3K27ac, BRD4L, and p300 at the same promoter site over the empty vector control whereas the latter is not (Fig. 6e). These results indicated that the TAD1/2 depletion undermines BRD4-NUT's ability to recruit p300 to ALX1 gene locus, resulting in reduced histone acetylation and ALX1 transcription.
To further investigate the role of BRD4-NUT in cell invasion and migration, we examined key EMT markers HEK293T cells transfected with wild-type BRD4-NUT or TAD1/2 deletion mutant. We found that ectopic expression of wild-type BRD4-NUT resulted in upregulation of gene transcription and protein expression of mesenchymal cell markers (Snail, vimentin and N-cadherin) and downregulation of epithelial marker E-cadherin, whereas the TAD1/2 deletion mutant had little effects (Fig. 7a, b), highlighting the importance of BRD4-NUT/p300 binding in EMT and potential epithelial morphology changes. This finding was supported by a wound-healing assay in HEK293T cells, which showed that wild-type BRD4-NUT but not the TAD1/2 deletion mutant substantially promoted cell migration capability as compared to the control cells (Fig. 7c). Finally, to directly investigate the transcriptional control of ALX1 by BRD4-NUT/p300 in NC cells, we performed siRNA knockdown of ALX1 and BRD4-NUT in NC 14169 cells, and found that siNUT reduced the transcription of ALX1, and both siNUT and siALX1 resulted in increased expression of Involucrin, a marker for differentiation (Fig. 7d, e), which is consistent with a previous study of NC TC-797 cells 48 . Collectively, these results highlighted that BRD4-NUT/p300 bipartite interaction is essential for transcriptional activation of ALX1 in NC cells.

Discussion
The identifications of recurrent gene fusions and their associated disease mechanisms in hematological disorders have facilitated the development of clinical biomarkers and new drugs targeting these fusion proteins. However, our current mechanistic understanding of fusion proteins in tumorigenesis of solid tumors is still limited. The BRD4-NUT fusion protein, a strong driver mutation in the poorly differentiated and highly aggressive NUT carcinoma 9 , offers an ideal model to study the fundamental structural and molecular principles of gene fusion activities in aberrant gene transcription in chromatin in solid tumors 9,49 . In this study, we discovered that the NUT moiety in BRD4-NUT contains two TADs that adopt amphipathic helices when bound to the TAZ2 domain of p300. The BRD4-NUT bipartite binding to p300 allosterically activates p300 by relieving TAZ2 autoinhibitory function on the HAT activity, resulting in trans-autoacetylation of p300. This p300 activation by NUT TADs/TAZ2 binding is similar to that of p53 tandem TADs interactions with p300 as shown in our study, and also reminiscent of p300 TAZ2 domain binding to dimerized transcription factors such as STAT1 and IRF3 30,31 . Notably, the importance of the autoinhibitory function of the TAZ2 domain on p300 HAT activity is underscored by the recent reports that TAZ2 deletion mutants of p300/CBP found in many human cancers are associated with histone hyperacetylation at H3K27 and H3K18 in chromatin, two known substrates for p300/CBP 9 , and recurrence following radiation in squamous cell carcinoma cohorts 50 .
BRD4-NUT/p300 forms discrete nuclear condensates in chromatin through multivalent molecular interactions involving the intrinsically disordered regions in BRD4, NUT, and p300, as well as BRD4 bromodomains' binding to acetylated histones and DNA in chromatin [37][38][39] . Within these condensates, BRD4-NUT/p300 are colocalized with H3K27ac and BRD4L/S, MED1, CDK9 and RNA PolII-S2P/ S5P, a complex highly competent for gene transcriptional activation. Importantly, our findings provide mechanistic details to a model in which BRD4-NUT recruitment of p300 through bipartite NUT TAD interaction with p300 TAZ2 domain triggers robust p300 HAT activation and resulting enrichment of H3K27ac in chromatin (Fig. 7f), which in turn recruits more BRD4-NUT and p300, nucleating a feedforward loop that propagates a wide spread of BRD4-NUT/p300 nuclear condensation-assisted unusually large topological domain structure (megadomain) in chromatin. The BRD4-NUT/p300 megadomain structure is likely confined by factors including major topologically associated domains (TADs) and competition between limited HATs and HDACs in large excess in the necleus 51 . Such large-scale alternations of chromatin landscape can result in aberrant gene transcription by sequestration of limited p300 to the highly concentrated transcriptional hub that favors activation of anti-differentiation genes for perpetual tumor cell growth in NC carcinoma.
NC is a highly aggressive solid tumor with limited treatment options other than surgery and chemotherapy 9,12,16 . BET BrD inhibitors have been evaluated in human clinical studies and shown initial efficacy for NC treatment [52][53][54][55] . However, NC patients develop drug resistance and relapse during the treatment 56,57 , prompting an urgent need for new therapeutic strategies. Our mechanistic insights from this study suggest directions in drug development targeting BRD4-NUT/ p300. We showed that spatially constrained inhibition of BRD4 tandem BrDs by bivalent inhibitor MS645 40 is much more effective than monovalent inhibitor JQ1 and HDAC inhibitors in disrupting BRD4-NUT/p300 nuclear condensation in chromatin. In addition, given the role of the BRD4-NUT TAD/TAZ2 binding in p300 recruitment and activation, chemical inhibition of the TAZ2 domain may be exploited to block BRD4-NUT/p300 interaction and to inhibit p300 activation and histone hyperacetylation required for tumorigenesis of NCs.
Pathological findings indicate that NCs resemble those of squamous cancer progression 41,47 , but how BRD4-NUT fusion protein drives NC invasion and metastasis is insufficiently understood. ALX1 is a homeodomain-containing transcription factor, and recently implicated as a regulator of Snail expression in activating cancer invasion and migration [43][44][45] . In NCs, excessive histone hyperacetylation domains enclose the ALX1 gene region 14 , and possibly change its expression as well as cellular functions controlled by ALX1. In this study, we showed that BRD4-NUT occupies at the promoter of ALX1, promotes coenrichment of p300, H3K27ac and BRD4L at its loci and activation of its transcriptional expression. Overexpression of BRD4-NUT is causally correlated with the upregulation of signature genes that favor the EMT signaling pathway and tumorigenesis of NC, whereas depletion of BRD4-NUT/p300 binding reduces ALX1 expression and consequently results in EMT signaling suppression. Our study suggests that the BRD4-NUT/p300 bipartite interaction likely plays an important role for propagating histone hyperacetylation, chromatin condensation and sustaining aberrant gene transcription to perpetual tumor cell growth in NC carcinoma.

Protein purification
TAZ2, TAZ2-NUT fusion protein and TAZ2-cherry protein expression were performed in BL21 (DE3) pLysS cells in TB medium supplemented with 100 μM ZnCl 2 , 2 g/L glucose for 16 h at 22°C. Labeled protein expression was performed in M9 medium with the addition of 1 g/L of 15 NH 4 Cl or 2 g/L 13 C-glucose. Purification was performed as previously described 29 .

GST-NUT fragments
Protein expression was performed in BL21 (DE3) pLysS cells in TB medium for 16 h at 16°C. Cells were collected and resuspended in cold lysis buffer (1 × PBS, 1 mM DTT, pH 7.4) under addition of protease inhibitor (Roche). After sonication and cell debris removal by highspeed centrifugation (23,447 × g), the cell lysate was loaded onto GST column. The protein was eluted with elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). Then, the protein was purified by size exclusion chromatography using Superdex 75 or 200 columns (GE Healthcare). The protein was concentrated by centrifugation (2240 × g) (Amicon Centrifugal Filter 10 kDa, MWCO), and concentrations were measured by UV absorbance at 280 nm and using the calculated extinction coefficient. The size and purity of the purified proteins were monitored by SDS-PAGE.

Pull-down experiments
For recombinant protein pull down assays, 10-20 μg of each purified protein in pull-down buffer (1 × PBS, 1 mM DTT, 1 mM PMSF, pH 7.4) was combined and incubated with 25 μL GST affinity resin for 1 h at 4°C under constant mixing. The resin was washed for four times with the same buffer and the complexes were eluted off the beads using elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). The elutes were clarified by centrifugation 10 min 18,800 × g, supernatant and input samples were collected for western blot. GST fusion proteins were separated by SDS-PAGE and visualized using Coomassie blue staining.
For cell lysate GST pull-down assay, HEK293T cells were transfected with pcDNA3.1-p300, and cells were collected after 48 h. 500 μg GST or GST-NUT (aa 300-700) protein was incubated with 50 μL GST beads at 4°C for 3 h in binding buffer (1×PBS, pH 7.4, 1 mM DTT, 1 mM PMSF). The beads were washed four times with binding buffer and incubated with the HEK293T cell lysates at 4°C overnight. The beads were washed four times in binding buffer and eluted with binding buffer containing 10 mM reduced glutathione. Samples were resolved on SDS-PAGE and processed for Western blotting.

Chemical shift perturbations and K D measurement
Chemical shift perturbation experiments were performed at 50 μM protein concentration and the same buffer and pH conditions as above. Each fragment was titrated in each protein at increasing concentration in a range between 7.8 μM and 1000 μM. As reference spectrum was used the free form. The weighted chemical shift difference (Δδ weighted ) was calculated using the equation: where ΔδH is the chemical shift on the proton and ΔδN is the chemical shift on the nitrogen which is scaled with a factor 0.15 to account for the different range of the amide proton and amide nitrogen. Chemical shifts for each backbone amide group were measured from the peak detected in apo form spectrum to the peak at the end of the titration. K D of binding for each fragment were estimated using the following equation: [P]t and [L]t are the total concentration of protein and ligand; Δδobs is the observed shift in regard to the reference, while Δδmax is the maximum shift obtained upon saturation and is extracted from the fitting. K D for each fragment was extrapolated as an average value of KD +/− S.E.M. of those resonances.

Structure calculations
Structures of the p300 TAZ2 domain/NUT peptide (aa 403-418) and TAZ2-NUT fusion protein were calculated with a distance-geometry simulated annealing protocol with CNS 60 . Initial protein structure calculations were performed with manually assigned NOE-derived distance constraints. hydrogen-bond distance, ϕ and ψ dihedral-angle restraints from the TALOS + (v3.70F1) prediction were added at later stage of structure calculations for residues with characteristic NOE patterns 61 . The converged structures were used for the iterative automated NOE assignment by ARIA refinement 62

Cell culture and transfection
The human embryonic kidney cell line HEk293T and human normal liver cell line LO2 were cultured in DMEM (with glutamine) containing 10% FBS (BI) and 100 μg/mL Penicillin/Streptomycin. Polyethyleneimine (Polysciences) transfection reagent was used for transient transfection in all of these cells.

RNA isolation, reverse transcription, and quantitative PCR
The total RNA was isolated and purified using the Trizol Reagent. 1 μg of total RNA was reverse-transcribed using the TransScript First-Strand Synthesis SuperMix Kit (TransGene, AT351). qPCR was run on a LightCycler® 480 instrument (Roche) using Fast SYBR Green Master Mix (Applied Biosystems, Cat#: 4385617). mRNA levels were determined by the relative standard curve method, normalized to GAPDH and presented as relative mRNA levels. qPCR analyses were done in triplicate (LightCycler 480 Software v1.5.1.62). Experiments were repeated at least twice. Primers are listed in Supplementary Table 3.

In vitro droplet assays
In vitro droplet assays were performed as previously described with slightly change 39 . Recombinant 6×His-EGFP-NUT (aa 300-700) was stored in 1×PBS, pH 7.4, and was supplemented with 360 mM NaCl. Recombinant 6× His-Cherry TAZ2 was stored in 25 mM Tris-HCl, pH 6.3, 200 mM NaCl, 25 μM ZnCl 2 . Protein was diluted in 1×PBS, pH7.4 buffer supplemented with indicated NaCl concentrations and 10% PEG-6000. The protein solution was loaded either into a 384-well plate or a homemade chamber comprising a glass slide with a coverslip attached by two parallel strips of tape.

Immunofluorescence staining
Cells were cultured on coverslips and fixed with 4% PFA for 10 min at room temperature. Cells were incubated in permeabilization buffer (0.1% Triton X-100) in PBS for 20 min at room temperature and blocked (3% BSA, 2% goat serum in PBS) for 60 min at room temperature. The following primary antibodies were used: Rabbit Monoclonal anti-Histone H3 (acetyl K18) (Abcam, Cambridge, UK) Fluorescence recovery after photo-bleaching (FRAP) imaging FRAP was performed on an upright Zeiss confocal microscope. Images were acquired using a ×63 oil-immersion objective at a zoom corresponding to 100 × 100-nm 2 pixel size, and the microscope was controlled using Zeiss Zen software. In most FRAP experiments, except where otherwise noted, 50 frames were acquired at 1 frame per second, allowing 3 frames to be acquired before the bleach pulse. A circular bleach spot was chosen in a region of homogenous fluorescence at a position at least 1 μm from the nucleus. The spot was bleached using maximal laser intensity and pixel dwell time corresponding to a total bleach time of~1 s.
Image analysis was performed using Image J software (ZEN 2012, Blue Edition). FRAP profiles were calculated using a ROI marked at the bleached area and use the plug-in FRAP profiler to obtain fluorescence intensity profiles. Fluorescence intensities in the ROI immediately after bleaching F(0) were subtracted from fluorescence intensities at all times F(t) and results were then normalized to pre-bleaching values. Results were then imported into Prism software for statistics analysis. We generally collected data from 3 cells per cell line per condition per day, and all presented data are from at least three independent replicates on different days. All data were pooled and fluorescence intensity at time points after the bleaching step were fitted to the equation: where F(t), F(-t) and F(0) are the average fluorescence of the ROI at any time, before bleaching and, immediately after bleaching, respectively. Mf is the mobile fraction, t 1/2 is the half time of recovery and t is time in seconds. In Prism, this fitting is achieved by using non-linear regression and the exponential one-phase association model using Y 0 = 0 and where Mf corresponds to the plateau value. We note that because the bleach duration was relatively long compared with the timescale of molecular diffusion, it is not possible to accurately estimate the immobile and free fractions from our FRAP curves.

Co-immunoprecipitation
HEK293T cells transfected with indicated plasmids for 48 h were lysed in the lysis buffer (20 mM HEPES, pH 7.9, 300 mM NaCl, 3 mM MgCl 2 , 0.1% NP-40) containing Protease Inhibitor Cocktail (Transgene, DI101-01) and 10 mM sodium butyrate for 30 min at 4°C with rotation. After a 20 min centrifugation at 18,800 × g at 4°C, the supernatants were collected and separated into input and immunoprecipitated (IP) lysates. IP lysates were mixed with a GFP specific antibody overnight at 4°C, and then were immunoprecipitated with 25 μl Dynabeads TM Protein G (pre-washed with PBS buffer three times, Invitrogen) for 4 h at 4°C. After washing with the same IP lysis buffer, IPs were released by adding 2× Protein Loading Buffer (TransGen Biotech) to beads and subjected to western blotting.

ChIP assay
For ChIP experiments, cells were cross-linked with formaldehyde before chromatin was extracted, sonicated, and incubated with primary antibodies  Table 4) relative to input levels after (IgG) background subtraction.

In vitro wound healing assay
Transfected cells were seeded on 6-well plates until grown to 80% confluency. The transfection efficiency is detected by the positive EGFP signal, all the transfection efficiency is over 90%. The protein expression level is also analysis by western blot and result is shown in Fig. 4e. Cells were scratched by use of a 200 μL pipette tip. After washed three times with PBS to remove debris, cells were cultured in FBS-free medium to block cell growth. Recovery from the scratched wound was monitored by the use of a microscope. Each migration experiment was carried out in triplicate and repeated for three times. The relative migration rate was calculated using ImageJ software (ImageJ 1.48 V). Distance migrated was calculated by subtracting the average distance between wound edges from that at the beginning.
Gene knockdown by siRNA NC 14169 cells were seeded to a density of 2 × 10 5 cells/well in a six-well plate. siRNA mixtures against Myc, Alx1 and Nutm1 were obtained from Dharmacon and prepared by diluting siRNA in serum-free transfection medium and mixed with DharmaFECT#1 diluted in serum-free transfection medium. The NC 14169 cells were then treated with the siRNA mixtures for 24 and 72 h, and cells were collected for qPCR and western blot analysis of mRNA and protein expression levels, respectively.

Analytical size exclusion chromatography
Purified MBP-TAZ2, His-F1c (aa 346-592) or MBP-TAZ2/His-F1c complexes (0.8 mM) were injected and eluted from the column (GE, Superdex 200 increase 10/300 GL) at a flow rate of 0.3 ml/min. The column was equilibrated with PBS buffer and calibrated using the Gel Filtration Markers Kit (Sigma, MWGF1000) comprising a set of molecular weight protein standards in the same buffer condition as indicated.
Droplet pelleting assay EGFP-NUT (aa 300-700, 10 μM) and TAZ2 (molar ratio 1:2) mixtures were subjected to in-vitro droplet assay. The complex samples were spun at 2500 × g for 5 min after post-incubation time as indicated. The supernatants (S) were collected and pellets (P) resuspended in PBS buffer. Both supernatants and pellets fractions were prepared in 4×Laemmli Sample Buffer (Biorad, #1610747) and heated to 60°C for 5 min prior to running SDS-PAGE. The resolved proteins were transferred to 0.22 μm PVDF membrane, immunoblotted with specific antibody, and revealed by an enhanced chemiluminescence method.

Statistics and reproducibility
Experimental data are presented as mean +/− S.E.M or S.D. Sample numbers are indicated in the relevant figure legends. All in vitro and cellular experiments were independently replicated at least twice and repeated at least three times within each of the experimental runs. Statistical significance calculations comparing two conditions were performed using a two-tailed unpaired Student's t-test (GraphPad Prism v6.0c and Excel for Mac v14.3.0) or two-sided p-values for each indicated comparison are derived from the R software loaded with Limma package (R software v3.2.3, Limma package v3.26.8), with p < 0.05 considered statistically significant unless stated otherwise. No statistical methods were used to predetermine sample size.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.